All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Coronary Artery Disease (like atherosclerosis) is the major cause of morbidity and mortality in the Western world and its pathogenesis involves complicated interactions between cells of the arterial wall, blood cells, and plasma lipoproteins [Ross R. (1993) Nature 362: 801-809; Glass C. K. and Witztum J. L. (2001) Cell 104:503-516]. Today, it is common knowledge that lowering cholesterol levels reduces the risk of heart attacks, strokes and other forms of atherosclerotic vascular disease. In addition, many recent studies have shown that oxidative stress is a mechanism with a central role in the pathogenesis of atherosclerosis, cancer, and other chronic diseases, e.g. diabetes. In this scenario, a key role is played by macrophages in the sub-endothelial space, which are activated by oxidized low-density lipoproteins (ox-LDL). Recently, endothelial dysfunction due to oxidative stress was identified as a priming factor in the course of the development of atherosclerotic plaques.
LDL-cholesterol (LDL-C) levels are currently recommended as the primary target for lipid lowering therapy for prevention of CVD. The role of LDL-C in the development of atherosclerosis, the relation between blood LDL-C levels and risk of CVD, and the beneficial effects of LDL-C lowering therapy are well established. Similarly, it is well known that low levels of HDL cholesterol (HDL-C) are associated with an increased risk for CVD independent of LDL-C levels, and that raising HDL-C has been shown to significantly lower CVD risk. Total cholesterol/HDL-C ratio is most predictive of CHD as was demonstrated in several retrospective clinical trials. However, other factors may significantly affect the risk for CHD, and there is a growing body of evidence strongly suggesting a role of plasma triglycerides concentrations as an independent risk factor of CHD [Assmann G., et al. (1996) Am. J. Cardiol. 77(14):1179-1184]. Omega-3 fatty acids influence CVD risk in a multifactorial manner. Numerous findings, including evidence from randomized controlled trials, demonstrated the beneficial effects of Omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) on CVD risk in patients with preexisting CVD as well as in healthy individuals. Large-scale epidemiological studies suggest that individuals at risk for CHD benefit from the consumption of plant- and marine derived Omega-3 fatty acids. Evidence from prospective secondary prevention studies and randomized controlled trials indicate that Omega-3 fatty acids supplements can reduce cardiac events (e.g., death, non-fatal myocardial infarct, non-fatal stroke) and decrease progression of atherosclerosis in coronary patients [Kris-Etherton P M., et al. (2002) Circulation 106(21):2747-2757] Omega-3 fatty acids reduce very-low density lipoprotein (VLDL) secretion, lower triglycerides transport and enhance VLDL clearance, and reduce circulating triglycerides. Omega-3 fatty acids have markedly anti-inflammatory, anti-thrombotic and immuno-modulatory properties that may be beneficial in CVD.
EPA and DHA may alter HDL cholesterol subclasses. Increases in the HDL2 subfraction have been reported with supplementation of 4 g DHA/d in hyperlipidemic men and type 2 diabetic patients [Mori, T. A. et al. (2000) Am. J. Clin. Nutr. 2000; 71: 1085-94]. The effect of EPA on HDL2 subclasses is less clear; a lowering effect on HDL3 concentrations, with no effect on HDL2 have been observed. When supplemented simultaneously, 1.48 mg DHA and 1.88 mg EPA/d were shown to increase HDL2 concentrations in subjects with familial combined hyperlipidemia, a disorder characterised by low HDL2 concentrations [Calabresi, L. et al. (2004) Metabolism 53:153-8]. HDL cholesterol concentrations are usually not significantly affected by plant sterols, but a slight increase has been reported in a few studies [Gylling, H. and Miettinen, T. A. (1999) Metabolism 48: 575-80].
Subjects identified as having low density lipoprotein (LDL) cholesterol concentrations above 130 mg/dl are routinely counselled to modify their diet with respect to saturated fat and cholesterol intake. At present the dietary guidelines are relatively broad; total fat 20-25% of energy (<10% of energy saturated fatty acids [SFA], 5-15% of energy monounsaturated fatty acids [MUFA], up to 10% of energy polyunsaturated fatty acids [PUFA], <300 mg cholesterol per day). In addition to the aforementioned dietary recommendations, the use of plant sterols and stanols to optimize blood lipid levels has gained increased importance through the year 2001, with the new recommendation released from the National Cholesterol Education Program advising the public to consume 2 g per day of plant sterols or stanols in addition to the Therapeutic Lifestyle Change Diet to lower elevated LDL cholesterol levels. Plant sterols and stanols are now widely available in many countries across the world as functional foods possessing government-approved health claims.
Paraoxonase (PON1) is an esterase, transported in the plasma as a component of HDL, associated to ApoAI and ApoJ. It has been shown in vitro that purified PON1 and HDL-associated PON1 inhibit the oxidative modification of LDL. Thus, the presence of PON1 in HDL may account for a proportion of the anti-oxidant properties of these lipoproteins [Tsuzura, S., et al. (2004) Metabolism. 53:297-302]. Interestingly, several investigators have shown that serum paraoxonase activity is lower in diabetic patients and is lower yet in those with diabetic complications, independent of PON1 gene polymorphisms. These observations are consistent with in vivo increased oxidative stress levels in diabetic patients.
The LDL oxidation hypothesis of atherosclerosis raised an extensive investigation into the role of anti-oxidants against LDL oxidation as a possible preventive treatment of atherosclerosis. Although increased resistance of LDL to oxidation was observed after treatment with various synthetic pharmaceutical agents, an effort has been made to identify natural food products, which offer anti-oxidant defense against LDL oxidation.
Olive oil has been shown to inhibit LDL oxidation and this effect could be related to its high oleic acid content, as well as to some phenolics (hydroxytoluene, oleoropein) and phytosterols such as sitosterol [Aviram M. and Kasem E. (1993) Ann. Nutr. Metabol. 37:75-84; Visioli F. et al. (1995) Atherosclerosis 117:25-32].
In addition to LDL oxidation, a known risk factor for coronary heart disease (CHD)—the result of atherosclerosis in the coronary arteries—includes high serum LDL cholesterol concentration. There is a positive linear relationship between serum total cholesterol and LDL cholesterol concentrations, and risk of, or mortality from CHD [Jousilahtu et al. (1998) Circulation 97:1084-1094]. High concentrations of serum triacylglycerols may also contribute to CHD [Austin, M. A. (1989) Am. J. Epidemiol. 129:249-259], but the evidence is less clear. Diacylglycerols (DAG) have been shown to lower the postprandial elevation of serum triacylglycerols levels compared with triacylglycerols in healthy humans [Taguchi, H et al. (2000) J. Am. Coll. Nutr. 19:789-796].
Phytosterols and CHD
The term “phytosterols” covers plant sterols and plant stanols, including beta-sitosterol, campesterol and stigmasterol. Plant sterols are naturally occurring substances present in the diet as minor components of vegetable oils. Plant sterols have a role in plants similar to that of cholesterol in mammals, e.g. forming cell membrane structures. In human nutrition, both plant sterols and plant stanols are effective in lowering total plasma cholesterol levels and LDL-cholesterol.
The consumption of plant sterols and plant stanols lowers blood cholesterol levels by inhibiting the absorption of dietary and endogenously-produced cholesterol from the small intestine. The plant sterols/stanols are very poorly absorbable compounds. This inhibition is related to the similarity in physico-chemical properties of plant sterols and stanols to cholesterol.
In addition, both plant sterols and plant stanols have been subjected to rigorous toxicological evaluation. Studies on the absorption, distribution, metabolism and excretion have shown that plant sterols are poorly absorbed from the intestine (1-10%).
The specific plant sterols that are currently incorporated into foods for their hypocholesterolemic effects are extracts of soybean or tall (pine tree) oils. In most cases these plant sterols are esterified to unsaturated fatty acids (creating sterol esters) to increase miscibility within the foods they are normally matrixed into. Some plant sterols currently in use are hydrogenated prior to esterification, resulting in saturated stanol derivatives, or plant stanols, such as beta-sitostanol and campestanol. Additional sources of plant sterols that are now becoming available are derived directly from corn fiber and not further modified, microcrystallinized in a way that obviates the need for esterification, or esterified to specific fatty acids that may have independent biological activity such as omega-3 fatty acids.
In the early 1950's plant derived sterols were first observed to decrease serum cholesterol levels and were marketed by Eli Lilly as Cytellin™.
Plant sterols consumption at 2-3 g/day has been demonstrated to lower circulating LDL cholesterol levels by 10-15% in humans with hyperlipidemia, offering a useful dietary strategy to risk management for heart disease. The mechanism of action of plant sterols in lowering LDL levels remains to be fully explained, however, it has been demonstrated that plant sterols act by excluding dietary and biliary cholesterol from micelles in the intestine during the process of absorption. Plant sterols, due to their chemical structure, prevent cholesterol from entering the outer micellar zone, thereby restricting the passage of cholesterol from the intestinal lumen across into the mucosal cell of the intestinal wall. Recently, it has been demonstrated that certain types of compounds, such as ascorbic acid, attached to the hydroxyl group of plant sterols such as sitosterol and campesterol as esters, increase the efficacy of cholesterol lowering in animal experiments, likely through more aggressive exclusion of cholesterol from the micelle.
In the United States, a panel of independent experts has concluded that vegetable oil sterol esters, meeting appropriate food-grade specifications and produced by current good manufacturing practice (21 C.F.R. §182.1(b)), are safe for use as an ingredient in vegetable oil spreads, in amounts which do not exceed 20% of plant sterol esters. It was the Panel's opinion, together with qualified experts in the field, that vegetable oil sterol esters are safe for use, i.e. vegetable oil sterol esters were granted the GRAS status (Generally Recognized As Safe). Based on the GRAS recognition, the US Food and Drug Administration (FDA) has cleared to use a spread containing up to 20% of plant sterol esters and another one containing plant stanol ester. Similar approvals were given in different European countries as well as in Asia and Australia.
A recent review teaches that in recent years, with the growing interest in functional foods, the use of phytosterols for reducing serum cholesterol levels has gained considerable momentum [Stark, A. H. et al. (2002) Nutrition Reviews 60(6):170-176]. This should be attributed, inter alia, to the esterification of phytostanol with fatty acids (stanyl esters), providing commercial scale production of phytosterol-containing foods, such as margarines. Like stanyl esters, phytosteryl esters (steryl esters) have been shown in clinical studies to consistently lower serum LDL-cholesterol (LDL-C) levels (reducing by up to about 10% or more), with no change seen in HDL-cholesterol (HDL-C) values. The review suggests that properly formulated free phytosterols and stanols may be as effective as stanyl and steryl esters in lowering LDL-C levels in humans.
WO 01/32035 teaches olive oil-based products, based on especially higher grades of olive oils (such as virgin olive oils), comprising plant stanol esters and/or plant sterol esters.
U.S. Pat. No. 5,843,499 discloses oil extractable from corn fiber that contains ferulate esters (phytosterol esters which are esterified to ferulic acid), in particular sitostanyl ester, which has been shown to have cholesterol-lowering activity. It is mentioned that corn fiber oil (containing about 73% fat (triacylglycerol), 8% sterol (fatty acyl) esters, 4% free sterols, 6% diacylglycerols and 6% ferulate (sterol esters)) is used as an additive to supplementary food for reducing cholesterol level.
U.S. Pat. No. 6,326,050 discloses a composition consisting of oil or fat, a diacylglycerol, a free phytosterol and tocopherol, dissolved or dispersed in the oil or fat. This composition plays a role in lowering blood cholesterol of hypercholesterolemic individuals.
However, none of the above mentioned publications describes reduction of both cholesterol and triglycerides serum levels.
Olive oil, in contrast to other mentioned vegetable oils (such as corn fiber oil, table cooking oil, soybean oil, rapeseed oil, rice bran oil, and palm oil) is composed, inter alia, of 55 to 85% monounsaturated fatty acids (MUFA), in particular oleic acid, which contribute to the high nutritional value of this oil. There are some distinct advantages of using olive oil over other vegetable oils. Diets rich in olive oil have been shown to be more effective in lowering total cholesterol and LDL-cholesterol than conventional dietary treatments not containing high levels of MUFA [Brown M. S and Goldstein J. L. (1983) Ann. Rev. Biochem. 52:223-261].
Furthermore, olive oil is an integral ingredient of the Mediterranean diet and accumulating data suggests that it may have health benefits that include reduction of risk factors of coronary artery disease, prevention of several types of cancer, and modification of immune and inflammatory response [Brown and Goldstein (1983) id ibid.].
WO01/15552 describes a nutritional supplement comprising purified esters of omega-3 fatty acids with phytosterols, for lowering triglyceride and cholesterol blood levels. This publication does not describe mixtures of such esters with a fat base, such as fish oil. Moreover, this publication does not describe any effect of the disclosed esters on blood lipids sub-fractions.
U.S. Pat. No. 6,589,588 discloses a sterol or stanol composition, wherein the fatty acid moiety comprises a blend of less that 7% of saturated fatty acids and more than 50% of polyunsaturated fatty acids, for lowering absorption of cholesterol for the digestive tract. Also this publication does not describe mixtures of the esters with fish oil, or any other fat, oil or lipid, and does not present any results as to the cholesterol reducing activity of the esters, neither any other activity, such as lowering triglycerides, controlling HDL subfractions, or the like.
Co-owned WO03/064444 describes a composition of matter comprising diacylglycerol(s), mainly 1,3-diacylglycerols (DAG) and phytosterol and/or phytostanol ester(s) (PSE), dispersed in oil and/or fat.
In the parent application, U.S. Ser. No. 11/199,584, the inventors report that said combination of diacylglycerol(s), mainly 1,3-DAGs, and PSEs, preferably dissolved or dispersed in oil and/or fat, has a synergistic effect and decreases both LDL-cholesterol and triglycerides levels in the blood. This combination further exhibited increased serum and macrophage anti-oxidative properties, and in particular LDL anti-oxidative properties, resulting in reduction of the risk for CHD and arteriovascular diseases.
In the parent application, an effect in reducing both cholesterol and triglycerides serum levels, together with increased anti-oxidative properties, was observed even when a combination containing only 11 wt % DAG and 20 wt % phytosterol esters (in oil) was employed.
It is an object of the present invention to provide mixtures of omega-3 fatty acids, preferably DHA and EPA, esterified to other lipids with improved activity in, e.g. reducing levels of as apolipoprotein B, decreasing levels of HDL subfraction HDL3, whilst elevating the level of HDL2.
These and other objects of the invention will become apparent as the description proceeds.